A consortium of productive biomedical investigators at Brown University and the University of Rhode Island is requesting support from the NCRR Shared Instrumentation Program for purchase of a mass spectrometer (MS) upgrade which would take an existing Thermo LTQ MS and transform it into a Thermo LTQ Orbitrap XL ETD hybrid MS. The resulting LTQ Orbitrap will be a high performance LC-MS and MSn system, combining rapid LTQ ion trap data acquisition with high mass accuracy Orbitrap mass analysis. Significantly, the Orbitrap XL ETD includes two fragmentation regimes, higher-energy C-trap dissociation (HCD) and electron transfer dissociation (ETD), that are complementary to the LTQ's collision induced dissociation (CID) option. These additional, alternative fragmentation capabilities are essential for the success of the diverse projects (phosphoproteomics;posttranslational modification analyses;"middle-down" proteomics) being actively pursued by the participating investigators. The requested new upgrade to the LTQ Orbitrap XL ETD MS is necessary for the collection and analysis of the high quality data required by the consortium's demanding and in some cases highly technically challenging research projects. The distinct benefits of the Orbitrap XL ETD mass spectrometer are its high sensitivity, resolution and mass accuracy, coupled to a fast scan rate. Most of the proposed major users participating in this proposal are pursuing the analysis of highly complex mixtures of proteins derived from cells or tissues. To adequately inventory the protein constituents within a complex proteomic sample, high quality data most be acquired on a time scale consistent with nano-liquid-chromatographic separation and elution. The Orbitrap is ideally positioned to provide the most robust, highest quality analysis of the complex samples described in this application. Once installed, this mass spectrometer will be utilized continuously (i.e., "24/7") through implementation of an in-house developed automated data pipeline which includes automated multidimensional capillary separations of peptides, nanospray LC/ESI-MS, automated data acquisition, and post-processing. Our current in-house technology platform provides for fully automated Sequest searching, quantitation of peptide abundance, statistical validation of database search results, and cross-referencing of observed peptides against an array of publicly available proteomic databases. Protein interactions and dynamics play a critical role in cell signaling, and form an important part of current efforts to explore and exploit the proteome. Systems network biology will be a vital component of our focus on proteins and their interactions in disease and development with the eventual goal of identification of new targets for drug therapy and the development of new approaches to therapeutics. As detailed in this proposal, such studies are presently an integral part of a number of existing and long-running NIH-sponsored research projects and with the recent investments in new faculty lines at Brown University in the Academic Enrichment Plan, the number and variety of proteomic studies will continue to grow over the next several years. It is very clear that the requested instrumentation will have an immediate and long-lasting impact in further propelling the high-quality biomedical research being pursued at Brown University and at the other institutions of higher learning in the state of Rhode Island. PUBLIC HEALTH RELEVANCE: The requested instrumentation upgrade will allow our participating investigators to elucidate the interacting proteins that are involved in the regulation of cell function and physiological response in a number of model systems ranging from asthma to cancer to neuronal signaling. These studies form an important part of current efforts to investigate and capitalize on our expanding knowledge of the proteome. This focus on proteins and their interactions in disease and development is being pursued with the eventual goal of identification of new targets for drug therapy and the development of new approaches to therapeutics. The basic research that will be facilitated by the requested instrumentation will greatly expand our knowledge of the interplay and cross-talk between various signaling pathways both in normal cellular and tissue function and in a variety of human disease states.